Aqueous cholesterol standard solution

ABSTRACT

Highly stable cholesterol standard solution comprising a known amount of cholesterol together with 1 to 20 volume percent of a primary or secondary aliphatic alcohol containing up to 4 carbon atoms; 5 to 20 volume percent hydroxy-polyethoxydodecane or 40 to 60 volume percent diethyleneglycol monobutyl ether; and 0.5 to 5 weight percent sodium chloride and/or 0.01 to 0.5 weight percent alkyl dimethyl benzyl ammonium chloride with 8 to 18 carbon atoms in the alkyl radicals.

United States Patent Stary et al.

[ June 24, 1975 AQUEOUS CHOLESTEROL STANDARD SOLUTION Inventors: Emil Stary, Hamburg; Peter Riieschlau, Tutzing, Obb; Erich Bernt, Munich, all of Germany Boehringer Mannheim Gmbll, Mannheim-Waldhof, Germany Filed: Apr. 30, 1974 Appl. No.: 465,640

Assignee:

Foreign Application Priority Data May 14. 1973 Germany 2324386 US. Cl. 252/408; 23/230 8; l95/l03.5 R Int. CL. G01n 33/16 Field of Search 23/230 B; 252/408 References Cited UNITED STATES PATENTS ll/l969 Cardinal 23/230 B 3,751,38l 8/1973 Megraw 23/230 B X 3,838,065 9/]974 Lippert et al. 252/408 FOREIGN PATENTS OR APPLICATIONS 1064,1533 4/1967 United Kingdom 23/230 B Primary Examiner-Joseph Scovronek Attorney, Agent, or Firm-Burgess, Dinklage & Sprung 5 7 1 ABSTRACT 22 Claims, No Drawings AQUEOUS CHOLESTEROL STANDARD SOLUTION The present invention relates to a cholesterol standard solution, more specifically, an aqueous solution of cholesterol with a definite and stable content of cholesterol.

A number of chemical and enzymatic processes are known or have been suggested for the quantitative determination of cholesterol in various materials, especially in biological fluids. In the case of these processes, in order to evaluate the measurement results, it is necessary to use cholesterol solutions with a definite, known content of cholesterol as standard. For this purpose, cholesterol solutions are used with a definite cholesterol content which are called cholesterol standards. In the case of the previously used standards, the cholesterol is dissolved in glacial acetic acid, chloroform, absolute alcohol or the like. However, the anhydrous standard solution so obtained gives, in the case of a cholesterol determination with the Liebermann- Burchardt color reagent, a color intensity which is different from that obtained with serum and can result in false cholesterol values in the serum (see L. Vass, Schweiz. med. Wschr., 102 914/1972).

This disadvantage can be overcome by the use of an aqueous cholesterol standard solution. However, the known aqueous cholesterol standard solutions, which contain hydroxypolyethoxydodecane as solubilizing agent, in turn suffer from a serious disadvantage insofar as the cholesterol content thereof changes rapidly and decreases noticeably even in the course of a few days. However, since it is, in the case of a cholesterol standard solution, especially important that the cholesterol content thereof remains absolutely constant, it was hitherto necessary freshly to prepare the aqueous cholesterol standard solution before use, which was a considerable disadvantage and, in addition, rendered questionable the dependability of the cholesterol determination.

The present invention obviates or mitigates these disadvantages and provides a stable, aqueous cholesterol standard solution.

The present invention provides an aqueous cholesterol standard solution, comprising, in aqueous solution, a known amount of cholesterol, together with 1 to 20 volume percent of a primary or secondary aliphatic alcohol containing up to 4 carbon atoms (for example, ethanol, isopropanol or n-butanol); 5 to 20 volume percent of hydroxy-polyethoxydodecane or 40 to 60 volume percent diethyleneglycol monobutyl ether; and 0.5 to 5 weight percent sodium chloride and/or 0.01 to 0.5 weight percent alkyl dimethyl-benzyl ammonium chloride with 8 to 18 carbon atoms in the alkyl radicals.

The present invention rests on the surprising discovery that a solution of cholesterol in dilute aqueous alcohol which, as solubilizing agent, contains the given amount of hydroxy-polyethoxydodecane or diethyleneglycol monobutyl ether, and, as stabilizing agent, sodium chloride and/or alkyl dimethylbenzyl ammonium chloride, is outstandingly stable and, even when stored for a year or more under normal conditions, does not change with regard to the cholesterol content.

A preferred cholesterol standard solution according to the present invention contains the dilute alcohol, hydroxy-polyethoxydodecane and sodium chloride. in this preferred composition, the best results have been obtained with a content of 8 to l2 vol. percent of an alcohol, for example ethanol, isopropanol or n-butanol, and 0.8 to L0 wt. percent sodium chloride. If, instead of sodium chloride, alkyl dimethyl-benzyl ammonium chloride is used, then the preferred amount is 0.05 to 0.15 wt. percent.

Another preferred cholesterol standard solution according to the present invention comprises 8 to 12 vol. percent of an alcohol, 55 to 60 vol. percent diethylene glycol monobutyl ether and 0.05 to 0.15 wt. percent alkyl dimethyl-benzyl ammonium chloride.

Yet another preferred cholesterol standard solution according to the present invention additionally contains a small amount of tetramethylurea. The amount thereof can be 0.5 to 5 vol. percent and is preferably 0.8 to 2.5 vol. percent.

Furthermore, depending upon the expected storage conditions, it can be expedient, for the prevention of bacterial attack, to add an alkali metal azide. In this case, the pH value must be adjusted to more than 6.5 by the addition of an appropriate buffer. It is preferred to add 0.05 to 0.2 percent and more, preferably about 0.1 percent of an alkali metal azide, for example sodium azide, in a buffer of pH 6.5 to 8, preferably of 7 to 8, for example, a triethanolamine buffer.

The cholesterol standard solution according to the present invention can contain up to 400 mg cholesterol in 100 ml of the solvent mixture. The extraordinary storage stability of the solutions necessitates neither the exclusion of light nor the exclusion of oxygen. This is all the more surprising since, as is known, cholesterol, even in a solid state, must be stored under an inert gas and with the exclusion of light. The stabilizing effect clearly depends upon the interaction of the various components of the standard solution according to the present invention since, with the individual components alone, a stabilizing effect cannot be achieved. For example, when a solution which originally contains 400 mg cholesterol in 100 ml of a mixture of 10 vol. percent hydroxy-polyethoxydodecane and vol. percent water, is stored for 3 weeks at 33C, only 80 percent of the original cholesterol is still found to be present. In contradistinction thereto, in the case of the standard solution according to the present invention in its various forms, even after storage for three months under the same conditions, no change of the cholesterol content can be ascertained. In the especially preferred embodiments of the present invention, complete stability was still retained after a period of storage of 2 years.

The following Examples are given for the purpose of illustrating the present invention:

EXAMPLE 1 40 g recrystallized cholesterol were dissolved in one liter hydroxypolyethoxydodecane by heating in a water-bath at 40C. This solution was diluted with doubly distilled water with a temperature of 40C, then ml of a 10 percent aqueous solution of alkyl dimethylbenzyl ammonium chloride and 1 liter ethanol were added thereto and, after cooling to ambient temperature, made up to precisely 10 liters with doubly distilled water.

EXAMPLE 2 40 g cholesterol were dissolved in 1 liter hydroxy-polyethoxydodecane and 1 liter ethanol and thereafter made up to liters with a 0.9 percent aqueous solution of sodium chloride.

EXAMPLE 3 40 g cholesterol were dissolved in 1 liter hydroxy-polyethoxydodecane, 1 liter n-butanol, 100ml of a 10 percent aqueous solution of alkyl dimethylbenzyl-ammonium chloride and 100 ml tetramethylurea and then made up to ID liters with doubly distilled water.

EXAMPLE 4 40 g cholesterol were dissolved in 6 liters diethyleneglycol monobutyl ether, 0.5 liters ethanol and 100 ml of a 10 percent aqueous solution of alkyl dimethylbenzyl-ammonium chloride were added thereto and then the mixture was made up to 10 liters with doubly distilled water.

It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. Cholesterol standard solution comprising in aqueous solution a. a defined amount of cholesterol;

b. l to volume percent, based on the standard solution, of a primary or secondary aliphatic alcohol of up to 4 carbon atoms;

c. 5 to 20 volume percent of hydroxy-polyethoxydodecane or 40 to 60 volume percent of diethyleneglycol monobutyl ether; and

d. at least one of i. 0.5 to 5 weight percent of sodium chloride; and ii. 0.01 to 0.5 weight percent of alkyl dimethyl benzyl ammonium chloride of from 8 to 18 carbon atoms in the alkyl moiety.

2. Standard solution as claimed in claim 1 wherein said primary or secondary aliphatic alcohol of component (b) is at least one of ethanol, isopropanol or nbutanol.

3. Standard solution as claimed in claim I wherein component (c) is hydroxy-polyethoxydodecane.

4. Standard solution as claimed in claim I wherein component (c) is diethyleneglycol monobutyl ether.

5. Standard solution as claimed in claim 1 wherein component (C!) is sodium chloride.

6. Standard solution as claimed in claim 1 wherein component (d) is alkyl dimethyl benzyl ammonium chloride.

7. Standard solution as claimed in claim 1 wherein component (d) is a combination of sodium chloride and alkyl dimethyl benzyl ammonium chloride.

8. Standard solution as claimed in claim 1 wherein component (b) is contained in an amount of from 8 to l2 volume percent of said solution.

9. Standard solution as claimed in claim 1 wherein component (c) is hydroxy-polyethoxydodecane and is contained in an amount of from 5 to 20 volume percent based on the standard solution.

10. Standard solution as claimed in claim 1 wherein component (d) is sodium chloride and is contained in an amount of from 0.8 to L0 weight percent.

11. Standard solution as claimed in claim 1 wherein component (d) is alkyl dimethyl benzyl ammonium chloride and is contained in an amount of from 0.05 to 0.15 weight percent.

12. Standard solution as claimed in claim 7 comprising 8 to 12 volume percent of said alcohol as component (b).

13. Standard solution as claimed in claim 1 comprising 55 to 60 volume percent of diethylene glycol monobutyl ether as component (c) and 0.05 to 0. l 5 weight percent of alkyl dimethyl benzyl ammonium chloride as component (d).

14. Standard solution as claimed in claim 1 additionally containing tetramethylurea.

15. Standard solution as claimed in claim 14 wherein the content of said tetramethylurea is from 0.5 to 5 volume percent based on said standard solution.

16. Standard solution as claimed in claim 15 wherein the content of said tetramethylurea is from 0.8 to L5 volume percent based on said standard solution.

17. Standard solution as claimed in claim 1 also con- 7 taining an alkali metal azide and wherein said solution is buffered to a pH of at least 6.5.

18. Standard solution as claimed in claim 17 wherein the content of said alkali metal azide is 0.05 to 0.2 percent by weight based on said standard solution.

19. Standard solution as claimed in claim 18 wherein the content of said alkali metal azide is about 0.1 weight percent.

20. Standard solution as claimed in claim 17 wherein said solution is buffered to a pH of 6.5 to 8.

21. Standard solution as claimed in claim 20 wherein said solution is buffered to a pH of 7 to 8.

22. Standard solution as claimed in claim 1 wherein the cholesterol content is up to 400 mg. per ml. of

said standard solution. 

1. CHOLESTEROL STANDARD SOLUTION COMPRISING IN AQUEOUS SOLUTION A. A DEFINED AMOUNT OF CHOLESTEROL; B. 1 TO 20 VOLUME PERCENT, BASED ON THE STANDARD SOLUTION, OF A PRIMARY OR SECONDARY ALIPHATIC ALCOHOL OF UP TO 4 CARBON ATOMS; C. 5 TO 20 VOLUMER PERCENT OF HYDROXY-POLYETHOXDODECANE OR 40 TO 60 VOLUME PERCENT OF DIETHYLENEGLYCOL MONOBUTYL ETHER; AND D. AT LEAST ONE OF I. 0.5 TO 5 WEIGHT PERCENT OF SODIUM CHOLRIDE; AND II. 0.001 TO 0.5 WEIGHT PERCENT OF ALKYL DIMETHYL BENZYL AMMONIUM CHLORIDE OF FROM 8 TO 18 CARBON ATOMS IN THE ALKYL MOIETY.
 2. Standard solution as claimed in claim 1 wherein said primary or secondary aliphatic alcohol of component (b) is at least one of ethanol, isopropanol or n-butanol.
 3. Standard solution as claimed in claim 1 wherein component (c) is hydroxy-polyethoxydodecane.
 4. Standard solution as claimed in claim 1 wherein component (c) is diethyleneglycol monobutyl ether.
 5. Standard solution as claimed in claim 1 wherein component (d) is sodium chloride.
 6. Standard solution as claimed in claim 1 wherein component (d) is alkyl dimethyl benzyl ammonium chloride.
 7. Standard solution as claimed in claim 1 wherein component (d) is a combination of sodium chloride and alkyl dimethyl benzyl ammonium chloride.
 8. Standard solution as claimed in claim 1 wherein component (b) is contained in an amount of from 8 to 12 volume percent of said solution.
 9. Standard solution as claimed in claim 1 wherein component (c) is hydroxy-polyethoxydodecane and is contained in an amount of from 5 to 20 volume percent based on the standard solution.
 10. Standard solution as claimed in claim 1 wherein component (d) is sodium chloride and is contained in an amount of from 0.8 to 1.0 weight percent.
 11. Standard solution as claimed in claim 1 wherein component (d) is alkyl dimethyl benzyl ammonium chloride and is contained in an amount of from 0.05 to 0.15 weight percent.
 12. Standard solution as claimed in claim 7 comprising 8 to 12 volume percent of said alcohol as component (b).
 13. Standard solution as claimed in claim 1 comprising 55 to 60 volume percent of diethylene glycol monobutyl ether as component (c) and 0.05 to 0.15 weight percent of alkyl dimethyl benzyl ammonium chloride as component (d).
 14. Standard solution as claimed in claim 1 additionally containing tetramethylurea.
 15. Standard solution as claimed in claim 14 wherein the content of said tetramethylurea is from 0.5 to 5 volume percent based on said standard solution.
 16. Standard solution as claimed in claim 15 wherein the content of said tetramethylurea is from 0.8 to 1.5 volume percent based on said standard solution.
 17. Standard solution as claimed in claim 1 also containing an alkali metal azide and wherein said solutIon is buffered to a pH of at least 6.5.
 18. Standard solution as claimed in claim 17 wherein the content of said alkali metal azide is 0.05 to 0.2 percent by weight based on said standard solution.
 19. Standard solution as claimed in claim 18 wherein the content of said alkali metal azide is about 0.1 weight percent.
 20. Standard solution as claimed in claim 17 wherein said solution is buffered to a pH of 6.5 to
 8. 21. Standard solution as claimed in claim 20 wherein said solution is buffered to a pH of 7 to
 8. 22. Standard solution as claimed in claim 1 wherein the cholesterol content is up to 400 mg. per 100 ml. of said standard solution. 